Various publications, including patents, published patent applications, technical or scholarly literatures and articles are cited throughout the present application. Each of these cited publications is incorporated by reference herein, in its entirety and for all purposes.
Opioid growth factor (OGF) is an endogenous opioid peptide ([Met5]-enkephalin) which belongs to a class of endogenous opioid proteins believed to be important in the growth of normal and neoplastic cells, and in renewing and healing tissues, in prokaryotes and eukaryotes (Zagon, I. S. et al., In: Cytokines: Stress and Immunity. Plotnikoff N P et al., (eds). CRC Press, Boca Raton, Fla., pp. 245-260, 1999).
OGF is directly involved in growth processes, and serves as a negative regulator in a wide variety of cells and tissues (Zagon, I. S. et al., In: Receptors in the Developing Nervous System. Vol. 1. Zagon, I. S. and McLaughlin, P. J. (eds). Chapman and Hall, London, pp. 39-62, 1993). OGF is known to modulate cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing and angiogenesis (Zagon et al., Brain Res 1999; 849(1-2):147-154).
OGF action is mediated by specific and saturable binding to a receptor called the OGF receptor or OGFr (Zagon et al., Brain Res Brain Res Rev. 2002 February; 38(3):351-76). OGFr has been cloned and sequenced in human, rat, and mouse and is characterized by containing a series of imperfect sequence repeats. Although the OGF receptor resembles classical opioid receptors pharmacologically, the OGF receptor has no resemblance to classical opioid receptors either at the nucleotide or protein levels. OGFr is associated with the outer nuclear envelope. The peptide-receptor complex associates with karyopherin, translocates through the nuclear pore, and can be observed in the inner nuclear matrix and at the periphery of heterochromatin of the nucleus, where OGF-OGFr signaling is thought to modulate DNA activity.
The interaction of OGF with OGFr to regulate cell replication serves as an active negative growth factor in neoplasia. As previously shown by Zagon et al. (Int. J. Oncology 17:1053-1061 (2000)), OGF acts on cell proliferation by targeting the G0/G1 phase of the cell cycle. OGF treatment resulted in a protraction of the G0/G1 phase, and therefore fewer cells enter mitosis and divide, resulting in the net effect of slowing growth progression.
The transition from one cell cycle phase to another is tightly regulated by different proteins that act at cell cycle control checkpoints. Key regulator proteins are the cyclin-dependent kinases (CDK), a family of serine/threonine kinases that are activated at specific points of the cell cycle. Thus far, there have been nine CDKs identified and, of these, five are active during the cell cycle. During G1 phase, CDK4, CDK6 and CDK2 are active. During S phase, CDK2 is active. During G2 and M phase, CDK1 is active.
CDK protein levels remain stable during the cell cycle, in contrast to their activating proteins, the cyclins. Cyclin protein levels rise and fall during the cell cycle and in this way they periodically activate CDK. Different cyclins are required at different phases of the cell cycle. The three D type cyclins (cyclin D1, cyclin D2 and cyclin D3) bind to CDK4 and CDK6 and CDK-cyclin D complexes are essential for entry into G1. Another G1 cyclin is cyclin E, which associates with CDK2 to regulate progression from G1 into S phase. Cyclin A binds with CDK2 and this complex is required during S phase. In G2 and early M phase, cyclin A complexes with CDK1 to promote entry into M. Mitosis is further regulated by cyclin B in complex with CDK1.
When activated, CDKs induce downstream processes by phosphorylating selected proteins. The most frequently studied target is the substrate of CDK4/6-cyclin D, i.e., the retinoblastoma (Rb) protein. During early G1, Rb becomes phosphorylated and this leads to disruption of a complex with various transcription factors, in particular, those belonging to the E2F family, which positively regulate the transcription of genes whose products are required for S phase progression (Nevins et al., Hum Mol Genet 10:699-703 (2001)).
Rb is one of the best characterized tumor suppressors and is recognized to participate in the regulation of the cell cycle, senescence, developmental processes, tissue homeostasis, and responses to chemotherapy. Its inactivation in almost every cancer and its intricate mode of operation and regulation has been widely reported (Liu et al., Curr Opin Genet Dev 14:55-64 (2004); Fan et al., Apoptosis 4:21-29 (1999)).
Rb belongs to a family of proteins known as “pocket proteins” (PP) which include Rb, p107 and p130 (Tonini et al., J Cell Physiol 192:138-150 (2002). Although all three protein are inactivated in various cancer cells, Rb emerged as the most pertinent tumor suppressor.
Rb suppressive activity is modulated by external stimuli that trigger the intracellular cascade of events that influence Rb interaction with E2F transcription factors. Hypo-phosphorylated Rb binds to E2F and suppresses E2F-dependent gene transcription. Phosphorylation releases Rb from E2F-bound promoters during the G1 (resting) phase of the cell cycle, leading to the accumulation of transcriptionally active E2F and activation of genes required for progression into the S-phase. Thus, the Rb phosophorylation state correlates with the well-characterized G1-S cell cycle restriction point.
Rb phosphorylation levels, in turn, are controlled by CDK kinases and CDK kinase inhibitors. CDK activity is governed, in part, by association with cell cycle inhibitory proteins called CDK inhibitors (CKI) which bind to CDK alone or to the CDK-cyclin complex and regulate CDK activity. Two distinct families of CKIs have been discovered, the INK4 family and Cip/Kip family, based on activity and sequence homology. The INK4 family includes p15 (INK4b), p16 (INK4a), p18 (INK4c), p19 (INK4d), which specifically inactivate G1 CDKs, i.e., CDK4 and CDK6. These CKI form stable complexes with the CDK enzyme before cyclin binding, preventing association with cyclin D. Inactivation of p16 is common in cancer in general and melanoma genesis in particular. The second family of inhibitors, the Cip/Kip family, includes p21 (Waf1, Cip1), p27 (Cip2), p57 (Kip2). These inhibitors inactivate G1 CDK-cyclin complexes (cyclin D-CDK4, cyclin D-CDK6 and cyclin E-CDK2) and to a lesser extent, CDK1-cyclin B complexes. Although p21 and p27 inhibit all CDK4/6 and CDK2 activity at high concentration, further analysis revealed that at physiological concentrations, cyclin E-CDK2 activity is inhibited whereas complex formation between CDK4/6 and cyclin D is augmented by p21 and p27, enhancing their kinase activity. This observation is in agreement with studies in which inactivating mutations in p16 facilitates immortalization of human melanocytes in culture but does not alter growth factor requirement. Likewise, mouse melanocytes with targeted disruptions in the p16, p21 or p27 became immortalized as their wild type counterparts and did not lose their dependency on external growth factor. However, disruption of p21 or p27, but not p16, accelerated melanocyte death in growth factor-deprived medium, suggesting that their presence protects the cells from apoptosis.
The underlying signaling mechanism by which OGF acts to negatively regulate cellular proliferation has been poorly understood. Currently, OGF is being used effectively as a chemotherapeutic agent, either alone or in combination with standard chemotherapies to treat a variety of cancers, including squamous cell carcinoma of the head and neck (SCCHN) and pancreatic adenocarcinoma. Although it is known that OGF acts on cell proliferation by targeting the G0/G1 phase of the cell cycle, the downstream effect or pathways that are involved have not been elucidated. Due to the critical role that the CDKs play in regulating cell cycle, a better understanding of the relationship between OGF and the CDK pathway is needed.
Therefore, there remains a need for a better understanding of the underlying mechanism of OGF's action and its relationship with the CDK pathway in regulating cell cycle. This understanding will provide improvements to the current methods of treating various types of cancers using OGF by enhancing the efficacy and responsiveness of current OGF therapy.
OGF is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. Its' action is mediated by the OGFr.
Previous studies have demonstrated the effectiveness of OGF as a useful agent in inhibiting tumor growth in a number of different tissues. Recent studies are directed at comparing the effectiveness of using either OGF alone or in combination with other chemotherapeutic agents in inhibiting tumor growth.
A major question in the use of OGF treatment or therapy has been identifying the eligibility of individuals who will be responsive to therapy with this agent. Moreover, the effectiveness of OGF therapy may be enhanced if other modalities were used in conjunction with this agent. A solution to these problems lies in understanding the underlying pathway of the OGF-OGFr axis in terms of influencing cell proliferation.